PROJECT SUMMARY This SBIR Phase II research program is focused on developing a small-molecule cancer immunotherapy drug that targets and activates the RIG-I like receptor (RLR) pathways, pattern-recognition receptors that function as intracellular sensor of RNA virus infection. Our small molecule agonists directly activate dendritic cells to prime an anti-tumor T cell response, and they induce immunogenic cell death (ICD) in cancer cells, a programmed cell death pathway that elicits an innate immune activation cascade within the tumor microenvironment. ICD primes a T cell response against tumor neoantigens and recruits activated T cells into the tumor. When administered by intratumoral injection, our lead series compounds, represented by the proof of concept example KIN1312, inhibit tumor growth in mice, and mice exhibiting complete tumor regression are immune to re-challenge with live tumor cells of the same type. In our Phase I studies, we used a structure-activity relationship (SAR) approach to identify KIN1312 analogs with improved drug-like properties, target binding, cellular potency, and efficacy. We obtained orthogonal confirmation of in vivo efficacy by demonstrating tumor regression in mouse CT26 and B16F10 tumor models, and we established that our agonists induce apoptosis in human cancer cell lines, but not in non-malignant primary cells. In addition, we demonstrated that combining KIN1312 with an anti-CTLA4 immune checkpoint inhibitor in the CT26 tumor model improved tumor regression and survival beyond that provided by either treatment alone. In Phase II, our goal is to select a lead preclinical candidate for advancement to investigational new drug (IND)-enabling studies as a new cancer immunotherapy. In Aim 1, we will use advanced SAR, structural biology, and formulation approaches to further optimize our innate immune agonists and to enable systemic administration. In Aim 2, we will determine the in vivo efficacy of these compounds in diverse tumor models and compare intratumoral and systemic routes of delivery. Compounds will be examined both as a monotherapy and in combination with immune checkpoint inhibitors and other immunotherapies. In Aim 3, we will define mechanisms of action and immune correlates of efficacy. High-priority compounds that exhibit synergistic activity with immune checkpoint inhibitors or other immunotherapies will be evaluated in toxicokinetic assays. Together, these studies will allow us to select intratumoral or systemic delivery for clinical proof of concept, and to select a lead preclinical candidate for advancement to IND-enabling studies. When used in combination with immune checkpoint inhibitors or engineered T cell therapies that target solid tumors, our RLR agonists have the potential to substantially expand the number of patients that could benefit from cancer immunotherapy.